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Casein kinase 2? regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

ABSTRACT: We have shown previously that the function of Ycf1p, yeast ortholog of multidrug resistance-associated protein 1 (MRP1), is regulated by yeast casein kinase 2? (Cka1p) via phosphorylation at Ser251. In this study, we explored whether casein kinase 2? (CK2?), the human homolog of Cka1p, regulates MRP1 by phosphorylation at the semiconserved site Thr249. Knockdown of CK2? in MCF7-derived cells expressing MRP1 [MRP1 CK2?(-)] resulted in increased doxorubicin sensitivity. MRP1-dependent transport of leukotriene C(4) and estradiol-17?-d-glucuronide into vesicles derived from MRP1 CK2?(-) cells was decreased compared with MRP1 vesicles. Moreover, mutation of Thr249 to alanine (MRP1-T249A) also resulted in decreased MRP1-dependent transport, whereas a phosphomimicking mutation (MRP1-T249E) led to dramatic increase in MRP1-dependent transport. Studies in tissue culture confirmed these findings, showing increased intracellular doxorubicin accumulation in MRP1 CK2?(-) and MRP1-T249A cells compared with MRP1 cells. Inhibition of CK2 kinase by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole resulted in increased doxorubicin accumulation in MRP1 cells, but not in MRP1 CK2?(-), MRP1-T249A, or MRP1-T249E cells, suggesting that CK2? regulates MRP1 function via phosphorylation of Thr249. Indeed, CK2? and MRP1 interact physically, and recombinant CK2 phosphorylates MRP1-derived peptide in vitro in a Thr249-dependent manner, whereas knockdown of CK2? results in decreased phosphorylation at MRP1-Thr249. The role of CK2 in regulating MRP1 was confirmed in other cancer cell lines where CK2 inhibition decreased MRP1-mediated efflux of doxorubicin and increased doxorubicin cytotoxicity. This study supports a model in which CK2? potentiates MRP1 function via direct phosphorylation of Thr249.

SUBMITTER: Stolarczyk EI

PROVIDER: S-EPMC3422697 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Casein kinase 2α regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

Stolarczyk Elzbieta I EI Reiling Cassandra J CJ Pickin Kerry A KA Coppage Ryan R Knecht Marc R MR Paumi Christian M CM

Molecular pharmacology 20120613 3

We have shown previously that the function of Ycf1p, yeast ortholog of multidrug resistance-associated protein 1 (MRP1), is regulated by yeast casein kinase 2α (Cka1p) via phosphorylation at Ser251. In this study, we explored whether casein kinase 2α (CK2α), the human homolog of Cka1p, regulates MRP1 by phosphorylation at the semiconserved site Thr249. Knockdown of CK2α in MCF7-derived cells expressing MRP1 [MRP1 CK2α(-)] resulted in increased doxorubicin sensitivity. MRP1-dependent transport of ...[more]

PMID: 22695718

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Project description:Malregulation of the actin cytoskeleton enhances tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin-related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in head and neck squamous cell carcinoma (HNSCC) and other cancers, corresponding with elevated tumor progression and poor patient outcome. Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. Although cortactin-mediated activation of Arp2/3 complex and invadopodia regulation has been well established, signaling pathways responsible for governing cortactin binding to Arp2/3 are unknown and potentially present a new avenue for anti-invasive therapeutic targeting. Here we identify casein kinase (CK) 2α phosphorylation of cortactin as a negative regulator of Arp2/3 binding. CK2α directly phosphorylates cortactin at a conserved threonine (T24) adjacent to the canonical Arp2/3 binding motif. Phosphorylation of cortactin T24 by CK2α impairs the ability of cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2α phosphorylation-null cortactin mutants, shRNA-mediated CK2α knockdown, and with the CK2α inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2α-mediated cortactin phosphorylation at T24 is critical in regulating cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. IMPLICATIONS: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.

Dataset Information

Casein kinase 2? regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

Publications

Casein kinase 2α regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

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